Tachypleus tridentatus Hemocyanin

Separation and Characterization of Monomer Subunits and Studies on Sulfhydryl Groups

Abstract

Hemocyanin monomers were isolated from Tachypleus tridentatus hemolymph by gel chromatography on Sephadex G-100. The isolated monomers were separated into four fractions by DEAE-Sephadex chromatography; they comprised 6 different subunits, designated as a-ξ chains. All of them showed the same molecular weight, 70, 000, on SDS-gel electrophoresis. The ξ chain was eluted ahead of the other subunits during gel chromatography. It tended to dimerize during prolonged dialysis or purification procedures. The a and ξ chains were isolated in pure form. The γ and δ chains, and also the β and ε chains, were obtained as mixtures but were not separated from each other. All the subunits showed different antigenicities. The amino-terminal portions of the α through ε chains have the same sequence, Thr-Ile•Leu-Lys-Glu-Lys-Gln. The ξ chain has a different amino-terminal sequence, Val-Leu-Asp-X-Ile/Leu-Glu-Lys. The ξ chain contained only 1 mol of Cu/mol of protein, whereas the other chains contained 2 mol of Cu/mol of protein. No free sulfhydryl group was detected in the absence of guanidine. However, 3 mol of SH/mol of protein was detected immediately after the addition of 6M guanidine-HCI. These SH groups were very unstable and disappeared on standing.